Advanced tissue engineering system

ABSTRACT

The invention is an automated advanced tissue engineering system that comprises a housing in which one or more tissue engineering modules are accommodated together with a central microprocessor that controls functioning of the tissue engineering modules. In one embodiment, the tissue engineering module comprises a housing supporting one or more bioreactor chamber assemblies and a fluid reservoir operationally engageable with the housing. The bioreactor chamber assemblies may be selected depending on the end product option desired and may include, for example, a cell therapy bioreactor chamber, a single implant bioreactor chamber and a multiple (mosaic) implant bioreactor chamber.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.17/513,457, filed Oct. 28, 2021, which is a continuation of U.S.application Ser. No. 17/013,100, filed Sep. 4, 2020, which is acontinuation of U.S. application Ser. No. 15/293,611, filed Oct. 14,2016, which is a divisional of U.S. application Ser. No. 11/597,550,filed Oct. 11, 2007, now U.S. Pat. No. 9,499,780, which is a U.S.National Phase under 35 U.S.C. § 371 of PCT/CA05/00783, filed May 26,2005, which claims benefit of U.S. Provisional Patent Application No.60/574,223, filed May 26, 2004, the disclosures of each of which areincorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The invention relates to a tissue engineering system. More specifically,the invention relates to an autologous advanced tissue engineeringsystem for automated cell therapy and tissue engineering for clinicalhospital settings.

BACKGROUND OF THE INVENTION

Different types of cell culture and tissue engineering devices have beendeveloped as are described for example in U.S. Pat. Nos. 5,688,687,5,792,603, 5,846,828, 5,994,129, 6,060,306, 6,048,721, 6,121,042,6,228,635 and 6,238,908. The major drawbacks of these devices are thefact that they have limited functional capabilities and are useful onlyfor the culture and expansion of cells. The devices are not designed forthe production of autologous tissue implants. Furthermore, these devicesare complex to use, bulky and thus not very portable, and still requireuser intervention in many aspects of the cell culturing process.

The Applicant has developed a fully automated tissue engineering systemdescribed in International Patent Application No. WO 03/0872292 (thedisclosure of which is incorporated herein in its entirety). The systemis a user-friendly and fully automated system for facilitating differentphysiological cellular functions and/or the generation of one or moretissue constructs from cell and/or tissue sources.

SUMMARY OF THE INVENTION

In one aspect, the present invention is an automated advanced tissueengineering system designed for further simplicity of use whilemaintaining aseptic conditions. The advanced tissue engineering systemcomprises a single housing operated by a central microprocessor unitthat holds one or more tissue engineering modules that can beindependently operated. The tissue engineering modules comprise ahousing having one or more chamber assemblies and, in aspects, comprisea tissue digestion chamber assembly, a proliferation chamber assemblyand a product chamber assembly. The product chamber assembly is selectedbased on the desired end use such as for cell collection for celltherapy or for various implant formation. The housing is operationallyengageable to a separate fluid reservoir that, in one aspect, snaps ontothe bottom of the housing. The tissue engineering module may also have avariety of biosensors to provide feedback with respect to the conditionswithin any of the chamber assemblies provided on the module as well asany fluids provided by the fluid reservoir and associated fluid tubing.Integrity sensors for monitoring that all parts are present andconnected together correctly on the module may also be incorporated.

The advanced tissue engineering system can bring turn-key productioncapability for autologous cell therapy and tissue engineering to ahospital clinic. The system can be designed for ease-of-use whilemaintaining aseptic conditions. The system can avoid the inherenthazards and cost related to the transportation and centralizedprocessing of human cells for various types of tissue repair and theadvanced system can also provide for autologous, rather than allogenicor xenogenic sources of cells, tissue or serum.

According to an aspect of the present invention is an advanced automatedtissue engineering system, the system comprising:

-   -   a housing;    -   one or more tissue engineering modules supported within the        housing; and    -   a central microprocessor that controls functioning of the one or        more tissue engineering modules.

According to an aspect of the present invention is an advanced automatedtissue engineering system, the system comprising:

-   -   a housing;    -   one or more tissue engineering modules supported within the        housing;    -   at least one biosensor associated with the housing and/or the        one or more tissue engineering modules; and    -   a central microprocessor that controls functioning of the one or        more tissue engineering modules.

According to another aspect of the present invention is a network ofautomated tissue engineering systems, wherein at least one of theautomated tissue engineering systems is the automated tissue engineeringsystem of present invention.

In aspects of the invention, the central microprocessor unit (CPU) ofthe system is used to programme and control the functioning andoperation of the entire system and the tissue engineering module(s)contained therein. For example, the CPU is used to release the tissueengineering module using an automatic sequence triggered by a usercommand on the touch screen display.

According to another aspect of the present invention is a tissueengineering module comprising:

-   -   a housing supporting at least one chamber assembly, the at least        one chamber assembly selected for at least one of tissue        digestion, cell proliferation, cell differentiation and implant        formation;    -   a fluid reservoir operationally engageable with the housing; and    -   at least one biosensor for the monitoring of parameters within        at least one of the fluid reservoir and the at least one chamber        assembly.

According to another aspect of the present invention is a tissueengineering module, the module comprising:

-   -   a housing supporting a number of chamber assemblies selected for        tissue digestion, cell proliferation, cell differentiation        and/or implant formation;    -   a fluid reservoir operationally engageable with the housing; and    -   at least one biosensor for the monitoring of parameters within        the chamber assemblies and/or within the fluid reservoir.

According to another aspect of the present invention is a tissueengineering module, the module comprising;

-   -   a housing supporting a tissue digestion chamber assembly, a        proliferation chamber assembly and a product chamber assembly;    -   a fluid reservoir connected to the housing and in fluid        communication with the tissue digestion chamber assembly, the        proliferation chamber assembly and the product chamber assembly;        and    -   at least one biosensor associated with one or more of the fluid        reservoir, the tissue digestion chamber assembly, the        proliferation chamber assembly and the product chamber assembly,        the at least one biosensor being in communication with a remote        central processor.

According to yet another aspect of the present invention is a tissueengineering module, the module comprising;

-   -   a housing, the housing supporting a removable tissue digestion        chamber assembly, a fixed proliferation bioreactor and a        removable product chamber assembly;    -   a fluid reservoir connected to the housing and in fluid        communication with the tissue digestion chamber assembly, the        proliferation bioreactor and the product chamber assembly; and    -   at least one biosensor associated with one or more of the fluid        reservoir, the tissue digestion chamber assembly, the        proliferation bioreactor and the product chamber assembly, the        at least one biosensor being in communication with a remote        central processor.

In aspects, the tissue engineering module is capable of conducting atleast one of tissue digestion, cell proliferation, cell differentiationand implant formation; individually, sequentially, predeterminedsequences or partial sequences.

In aspects, the product chamber assembly is configured depending on theend product option desired and may be selected to include for example acell therapy bioreactor that collects and holds proliferated cells forcell therapy applications; and a differentiation bioreactor for thedifferentiation of cells into either a single implant, multiple (mosaic)implant or a cell matrix implant.

According to yet another aspect of the present invention is a tissuedigestion chamber assembly, the assembly comprising:

-   -   a protective containment unit comprising a unit lid and a unit        base; and    -   a tissue digestion bioreactor within the protective containment        unit.

According to still another aspect of the present invention is a tissuedigestion chamber assembly, the assembly comprising:

-   -   a protective containment unit comprising a unit lid and unit        base; and    -   a tissue digestion bioreactor supported within the protective        containment unit.

In aspects, the tissue digestion chamber assembly is portable and can bemounted within a tissue engineering module. The tissue digestion chamberassembly is primarily for the digestion of patient biopsy material toretrieve cells for further proliferation, differentiation and/or implantformation. However, the tissue digestion chamber can also be used inaspects to directly receive patient cells without the need for anydigestion.

According to yet another aspect of the present invention is aproliferation chamber assembly, the assembly comprising:

-   -   a proliferation bioreactor comprising a proliferation chamber        having a base, a lid for containment of fluid, and a channel        system therein.

According to still another aspect of the present invention is aproliferation chamber assembly, the assembly comprising:

-   -   a proliferation bioreactor comprising a substantially flat base        with a large surface area, the base having a channel system        therein for flow of medium and cells; at least one biosensor to        detect and provide feedback on the condition of cell culture and        proliferative activity; and a lid to provide a chamber for        containment of fluid.

In aspects of the invention, the proliferation bioreactor is mountedwithin a housing of a tissue engineering module. This mounting may inaspects be fixed. The proliferation bioreactor may also in aspectscomprise a gas permeable membrane, flow interrupters, and vibratoryelements. In other aspects, the proliferation bioreactor may be providedhaving one or more bases stacked on top of one another to provideadditional surface area for the proliferation of cells. The base(s) mayalso be mounted within the bioreactor at an angle to provide anelevational change from inlet to outlet.

According to another aspect of the present invention is a productchamber assembly, the assembly comprising:

-   -   a protective containment unit comprising a unit lid and a unit        base; and    -   a product bioreactor within the protective containment unit.

According to yet another aspect of the present invention is a productchamber assembly, the assembly comprising:

-   -   a protective containment unit comprising a unit lid and a unit        base; and    -   a product bioreactor supported within the protective containment        unit.

According to still another aspect of the present invention is a productchamber assembly, the assembly comprising:

-   -   a protective containment unit comprising a unit lid and unit        base; and    -   a differentiation bioreactor supported within the protective        containment unit.

In aspects, the differentiation bioreactor is configured for thecollection of cells, for the generation of one or more implants, or forthe generation of cell matrix implant. In aspects of the invention, theproduct chamber assembly can be reversibly mounted to a tissueengineering module.

In other aspects of the invention, any one of the chamber assemblies canbe engageable with a tissue engineering module. This can be done in areversible manner or in an irreversible manner with any of theassemblies as desired. For example, it may be desirable in aspects tohave the tissue digestion chamber assembly non-removable after initialinstallation to ensure that the digest bioreactor is not re-used. Bydefault, this ensures that the remainder of the tissue engineeringmodule cannot be re-used.

It is noted that although the tissue engineering module is shown to havea tissue digestion chamber assembly, a proliferation chamber assemblyand a product chamber assembly, not all of these assemblies need to beused to create a final end product for clinical use. As a non-limitingexample, cells provided by enzymatic digestion of a surgical biopsy inthe tissue digestion chamber assembly can be moved either to theproliferation chamber assembly or directly to the product chamberassembly.

According to yet another aspect of the present invention is a tissueengineering module comprising at least one of the tissue digestionchamber assembly, the proliferation chamber assembly, and the productchamber assembly of described herein.

According to another aspect of the present invention is a tissueengineering module comprising:

-   -   a housing supporting a fluid reservoir and at least one of the        tissue digestion chamber assembly, the proliferation chamber        assembly, and the product chamber assembly of described herein;        and    -   at least one biosensor for the monitoring of parameters within        at least one of the fluid reservoir and said at least one        chamber assembly.

Other features and advantages of the present invention will becomeapparent from the following detailed description and drawings. It shouldbe understood, however, that the detailed description and drawing whileindicating embodiments of the invention are given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a perspective view of an advanced tissue engineering systemof one embodiment of the present invention;

FIG. 2 shows the advanced tissue engineering system of FIG. 1 with oneof the bay doors in the open position;

FIG. 3 shows a perspective view of one side of an embodiment of a tissueengineering module of the present invention removed from the bay door ofFIG. 2;

FIG. 4 shows a perspective view of the other side of the tissueengineering module of FIG. 3;

FIG. 5A shows a perspective view of one embodiment of a tissue digestionchamber assembly of the present invention;

FIG. 5B shows a partial exploded view of the tissue digestion bioreactorassembly of FIG. 5A showing a protective unit lid, a tissue digestionbioreactor, and a unit base;

FIG. 5C shows a partial exploded view of the tissue digestion bioreactorof FIG. 5B;

FIG. 6 shows a perspective view of an embodiment of a proliferationchamber assembly of the present invention;

FIG. 7A shows a perspective view of one embodiment of a product chamberassembly of the present invention;

FIG. 7B shows a partial exploded view of the product chamber assembly ofFIG. 7A showing a protective unit lid, a differentiation bioreactor, anda unit base;

FIG. 7C shows a partial exploded view of the differentiation bioreactorof FIG. 7B;

FIG. 8A a perspective view of another embodiment of a product chamberassembly of the present invention;

FIG. 8B shows a partial exploded view of the product chamber assembly ofFIG. 8A showing a protective unit lid, a cell therapy bioreactor, and aunit base;

FIG. 8C shows a partial exploded view of the cell therapy bioreactor ofFIG. 8B;

FIG. 9 shows a perspective view of an embodiment of a fluid reservoir ofthe present invention;

FIG. 10 shows a perspective view of an embodiment of a flow controlvalve housing of the tissue engineering module of FIG. 3;

FIG. 11 shows an exploded view of the tissue engineering module of FIG.4 with the fluid reservoir of FIG. 9;

FIG. 12 shows a scheme of a general methodology for clinical tissueengineering using the tissue engineering module of FIG. 3; and

FIG. 13 shows an embodiment of a fluid flow schematic.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is an improvement to the Applicant's automatedtissue engineering system described in International Patent ApplicationNo. WO 03/0872292 (the disclosure of which is incorporated herein in itsentirety). The advanced automated tissue engineering system of theinvention has a variety of improvements incorporated therein in order toincrease the ease-of-use, maintain the aseptic conditions of the systemand simplify the manufacture of the system, yet basically operates inthe same manner as the system of Applicant's International PatentApplication No. WO03/0872292.

The present invention will now be described in more detail withreference to the Figures. FIG. 1 shows the advanced tissue engineeringsystem 100. The system comprises a housing 102, multiple bays 106 eachaccommodating a tissue engineering module (not shown) and a userinterface touch screen 110. FIG. 2 shows the tissue engineering system100 with one of the bays 106 and the associated door in the openposition. The automated insertion and retraction system is supported bya guide rail system 120. Each of the bays can contain one tissueengineering module 118 which is secured to the bay by automated latchesresiding in the bay (not shown) and can be programmed to be removedthrough the entry of a specific password via the touch screen to preventunauthorized or unscheduled access or tampering.

FIGS. 3 and 4 show two side perspective views of the assembled tissueengineering module 118 removed from the bay. The module 118 has two maincomponents; the upper housing 200 and the lower fluid reservoir 300. Theupper housing 200 has a tissue digestion chamber assembly 500, aproliferation chamber assembly 600, and a product chamber assembly 700.There are fluid access ports 210 located on one side of the module 118and internally connected to the fluid pathway (not shown) within themodule 118, which can be used for quality control sample removal andcomponent addition to the internal fluid system if required. There areflow control valves 212 located on the other side of the module 118.

The chamber assemblies 500, 600 and 700, the lower fluid reservoir 300,and the remainder of the module 118 are each described below,respectively.

FIG. 5A shows the tissue digestion chamber assembly 500. FIGS. 5B and 5Cillustrate the components of the tissue digestion chamber assembly 500.The chamber assembly 500 comprises a tissue digestion bioreactor 510,described more fully with respect to FIG. 5C, and an outer protectivecontainment unit 520. The outer protective containment unit 520comprises a protective unit lid 522 and a unit base 524 to enhance theprotection and isolation of the contents of the bioreactor 510 therein.The tissue digestion bioreactor 510 is connected to the protective unitlid 522 via sterile needleless connectors 526. Each of the connectors526 is in direct fluid communion with a corresponding mating connector528 of the protective unit lid 522. Once the tissue digestion bioreactor510 is connected to the protective unit lid 522, the protective unit lid522 is then connected to the unit base 524.

The tissue digestion bioreactor 510 shown in FIG. 5C has four primarycomponents: a bioreactor base 530 that substantially forms a tissuedigestion chamber 532 of an appropriate size to accommodate one or moretissue samples such as a tissue biopsy (not shown); a removablebioreactor lid 534; a port filter 536, and an optional port filter (notshown).

The bioreactor lid 534 provides for sterile needleless connectors 526located at the ends of internal ports 538 and 540. The term “needlelessconnector” is understood to be a connector with no sharp needles (e.g. ablunt cannula). When the bioreactor lid 534 is assembled to thebioreactor base 530, internal port 538 is in fluid communion with thecentral tissue digestion chamber 532. Fluid may be transferred from port538 to and from chamber 532 across an optional port filter (not shown).Similarly, internal port 540 within the bioreactor lid 534 is in fluidcommunion with the bioreactor base 530, which in turn is in fluidcommunion with the tissue digestion chamber 532. Fluid may betransferred from port 540 to the tissue digestion chamber across theport filter 536. The role of the port filters 536 is to retain tissueaggregates and biopsy debris within the tissue digestion chamber 532while allowing the passage of disassociated cells out of the tissuedigestion chamber 532, via port 540.

Loading of a tissue biopsy into the tissue digestion chamber 532 isperformed with the bioreactor lid 534 removed from the bioreactor base530. Following loading, the lid 534 and base 530 are assembled togetherand the tissue digestion chamber 532 is operationally engaged with themodule 118 and then filled under automated control with an enzymesolution through port 540. The addition of enzyme solution to the tissuedigestion chamber 532 is balanced by air escaping through air vent 542.Biopsy digestion takes place under continuous or intermittentrecirculation of the enzyme solution, thereby keeping the released cellsin suspension and maximizing the exposure of the biopsy to the enzymereagents. During recirculation, the enzyme solution enters the bottom ofthe tissue digestion chamber 532 through the port filter 536 via port540 and leaves the top of chamber 532 via port 538. This creates a fluidflow path in a direction opposite to the gravity vector such that thebiopsy is suspended and tumbled to maximize the effectiveness of theenzyme reagents. Digestion may be enhanced by gentle agitation of thedigestion medium within the digestion chamber via a mixing diaphragm(not shown). The air vent 542 may be closed during any recirculationsteps, as any residual air bubbles present in the fluid flow system aretrapped and retained in the upper half of the bioreactor, above theinlet of port 538. Upon completion of the digestion sequence, theapplication of reverse flow of either air or medium via port 538 intothe top of the tissue digestion chamber 532 results in the dispensing ofthe disassociated cells past the port filter 536 and out of thebioreactor via port 540 to either a proliferation chamber assembly 600or a cell collection vessel. It is understood by one of skill in the artthat the tissue digestion bioreactor 500 can be optionally loaded withcells instead of a tissue, in this case digestion of the cells is notrequired.

Once the tissue digestion bioreactor 510 is assembled and placed withinthe outer protective containment unit 520, as shown in FIG. 5A, twinlayers of containment and protection are present for transport from theoperating room, where the biopsy is harvested, to the clinical areacontaining the system 100. Peel tape seals are present over the sterileneedleless connectors 528 such that the sterility of these connectors ismaintained until it is time to install this assembly 500 into the tissueengineering module 118.

FIG. 6 shows the proliferation chamber assembly 600. The proliferationchamber assembly 600 comprises a proliferation bioreactor 602 that has aproliferation chamber 604. The proliferation chamber 604 has a base 606having a proliferation surface 610 suitable for cell attachment andgrowth and a lid 611 for containment of fluid. To adjust or maintain thelevels of dissolved gases in the medium, a gas permeable membrane (notshown) may be incorporated to the top surface of the proliferationchamber 604 that allows the transport of gases such as oxygen and CO₂.Separation walls 612 divide the internal space of the proliferationchamber 604 into a channel system that forces medium to follow apredefined pathway from an inlet port 616 to an outlet port 618.

The design of the proliferation chamber assembly 600 has severalimportant operational features. Relatively uniform cell seeding can beobtained by the infusion of a cell suspension through the channelsystem. Furthermore, the channel configuration ensures that media flowis well distributed over the whole proliferation surface 610, therebyreducing potential low-flow regions that may compromise local cellvitality due to reduced nutritional supply or waste product removal.Confluence sensors 620 may be distributed through the chamber 604 toautomate the detection of final cell confluence. These sensors 620provide feedback on the progress of the cell culture activity tofacilitate automatic control over the entire process. In addition,information generated from the sensor data enables the operator toobtain advanced notification of processing status such that relatedclinical activities may be scheduled as appropriate.

At the conclusion of the proliferation sequence, continuous orintermittent recirculation of an appropriate enzyme solution through thechannel system induces cell detachment due to the effect of the enzymereaction and the low-level sheer stresses generated by the fluid flow.Accordingly, cell harvest is achieved without the need for mechanicalshaking or rotation of the proliferation chamber assembly 600.

The channel system used herein can provide for a uniform distribution ofcells to enable homogeneous cell feeding of the proliferation surface.

The inlet and outlet ports 616 and 618 connect with the proliferationchamber 604 via ducts (not shown) that increases in width as the base606 of the proliferation chamber 604 is approached. This reduces thestreamlining of the flow and allows a more uniform fluid distributioninto and out of the proliferation chamber 604.

The interior height of the proliferation chamber 604 within theproliferation chamber assembly 600 has been optimized to obtain anintermediate height between a low height that allows air bubbles tobridge between the lid 611 and the proliferation surface 610 (causingcell necrosis), and a high height where the fill volume is excessive andair removal is problematic.

The proliferation bioreactor 602 optionally includes flow interrupters(not shown) that deliberately cause controlled turbulence along thelength of the proliferation chamber 604. These interrupters are placedperpendicular to the flow as irregularities in the top surface of theproliferation chamber 604. The flow interrupters cause controlled mixingalong the length of the chamber 604 so that free cells (particularlypost release after confluence detection) remain in suspension and can bemoved efficiently toward the outlet 618.

The proliferation bioreactor 602 optionally includes a progressivechange in elevation along the length of the fluid pathway from inlet 616to outlet 618 to enable the more complete exhaust of all contents. Thiselevation change is accomplished with both the proliferation surface 610of the base 606 and the top surface of the chamber 604 changing inelevation at the same rate, thereby maintaining a consistent separationbetween the top surface and proliferation surface 610.

The proliferation bioreactor 602 optionally includes a vibratory element(not shown) that facilitates cell release from the proliferation surface610. This element is optionally mounted directly onto the chamber 604.

The proliferation chamber 604 optionally includes multiple bases 606(e.g. proliferation surfaces 610) in a stacked geometry whereby eachlevel is either in series or in parallel in terms of fluid flow.

FIG. 7A shows the product chamber assembly 700. FIGS. 7B and 7Cillustrate the components of the product chamber assembly 700. Thechamber assembly 700 comprises a differentiation bioreactor 710,described more fully with respect to FIG. 7C, and an outer protectivecontainment unit 720. The outer protective containment unit 720comprises a protective unit lid 722 and a unit base 724 to enhance theprotection and isolation of the contents of the differentiationbioreactor 710 therein. The differentiation bioreactor 710 is connectedto the protective unit lid 722 via sterile needleless connectors 726.Each of the connectors 726 is in direct fluid communion with acorresponding mating connector (not shown) of the protective unit lid722. Once the differentiation bioreactor 710 is connected to theprotective unit lid 722, the protective unit lid 722 is then connectedto the unit base 724. This forms twin layers of containment andprotection. Additionally, peel tape seals are present over the sterileneedleless connectors such that the sterility of these connectors ismaintained until it is time to install this assembly 700 into the tissueengineering module 118.

The differentiation bioreactor 710, designed to promote celldifferentiation and subsequent tissue construct formation, is shown inFIG. 7C. The differentiation bioreactor 710 has four primary components:a bioreactor base 730 that substantially forms a differentiation/tissueformation chamber 732; a removable bioreactor lid 734; a permeablemembrane tube 736; and a scaffold/membrane/matrix 738. The permeablemembrane tube 736 tightly encircles the scaffold/membrane/matrix 738 toform a cell and tissue growth compartment 740 above thescaffold/membrane/matrix 738. The tissue growth compartment 740 mayextend within the scaffold/membrane/matrix 738 according to the poresize of the scaffold/membrane/matrix 738 and the placement of thescaffold/membrane/matrix 738 within the membrane tube 736. The membranetube 736 is also affixed to the inlet port 742, such that the membraneis physically located within the differentiation/tissue formationchamber 732. This divides the bioreactor into two independentcompartments, a cell and tissue growth compartment 740 and an outercell-free medium compartment 744, all within chamber 732. The pore sizeof the membrane tube 736 is selected on the basis of being impermeablefor cells but permeable for nutrients, waste products, growth factors,etc., within the culture medium. If desired, membrane pore size can bechosen in a manner to exclude molecules of a certain molecular weightfrom passing through the membrane.

The inlet port 742 is required for loading a cell suspension into thetissue growth compartment 740 and for the perfusion of the emergingtissue construct with culture medium. During the delivery of the cellsuspension into the empty tissue growth compartment, entrapped airwithin the tissue growth compartment 740 is allowed to exit through airvent 746. In a similar fashion, the outer cell free compartment 744 ofchamber 732 is loaded with media via port 748 and entrapped air mayescape via air vent 750.

The design of the differentiation bioreactor 710 allows direct perfusionof the tissue construct through media delivery to port 742 or indirectmedia supply to the surrounding cell free compartment 744 of chamber 732via port 748. The indirect media supply is located away from that regionof the implantable scaffold/membrane/matrix 738 that is seeded withcells so as to minimize the potential for damaging sheer stresses thatcould compromise the formation of cell aggregates. Typically, ports 746and 750 are closed during perfusion and port 752 serves as a mediaoutlet; however, various alternate media supply scenarios are possiblebased on specific tissue engineering requirements or advanced cellculture requirements. An important aspect of the media perfusionstrategy is that the permeable membrane 736, which forms part of thetissue growth compartment 740, allows fresh culture medium to permeateinto the tissue growth compartment 740 without any loss of cells awayfrom the scaffold. Furthermore, nutrition is provided to the cells fromessentially all directions without restrictions from any impermeablebioreactor walls.

The differentiation bioreactor 710 complete with the protectivecontainment unit installed, as shown in FIG. 7A, represents thepre-assembled and sterilized format for the assembly 700. In use, thisassembly 700 is removed from the tissue engineering module 118 uponcompletion of the biological processing and is transferred to theoperating room. By virtue of the progressive layers of containment, theassembly is ideally suited for operating room aseptic procedures.

FIGS. 8A to 8C show the configuration of another embodiment of a productchamber assembly of the present invention. FIG. 8A shows a productchamber assembly 800. FIGS. 8B and 8C illustrate the components of theproduct chamber assembly 800. The chamber assembly 800 comprises a celltherapy bioreactor 810, described more fully with respect to FIG. 8C,and an outer protective containment unit 820. The outer protectivecontainment unit 820 comprises a protective unit lid 822 and a unit base824 to enhance the protection and isolation of the contents of the celltherapy bioreactor 810 therein. The cell therapy bioreactor 810 isconnected to the protective unit lid 822 via sterile needlelessconnectors 826. Each of the connectors 826 is in direct fluid communionwith a corresponding mating connector (not shown) of the protective unitlid 822. Once the cell therapy bioreactor 810 is connected to theprotective unit lid 822, the protective unit lid 822 is then connectedto the unit base 824. This forms twin layers of containment andprotection. Additionally, peel tape seals are present over the sterileneedleless connectors such that the sterility of these connectors ismaintained until it is time to install this assembly 800 into the tissueengineering module 118.

The cell therapy bioreactor 810 receives cells following proliferationand cell washing. The bioreactor 810 is shown in FIG. 8C. The celltherapy bioreactor 810 has a bioreactor lid 828 connected to a verticalchamber 830 and a bioreactor base 832 that contains a conical cellcollector 834. When the lid 828 is connected to the bioreactor base 832for operation in the module 118, a cell suspension may be introducedinto the bioreactor 810 and under quiescent conditions the cells settleby gravity into the conical cell collector 834. The combination of asealed bioreactor chamber shelf 836 and the fluid return tube 838 allowsthe remaining fluid above the conical cell collector 834 to be removedfrom the bioreactor 810 thus leaving concentrated cells within theconical cell collector 834 ready for implantation.

The cell therapy bioreactor 810 complete with the protective containmentunit installed, as shown in FIG. 8A, represents the pre-assembled andsterilized format for the assembly 800. In use, this assembly 800 isremoved from the tissue engineering module 118 upon completion of thebiological processing and is transferred to the operating room. Byvirtue of the progressive layers of containment, the assembly is ideallysuited for operating room aseptic procedures.

FIG. 9 shows the fluid reservoir 300. The internal elements of the fluidreservoir 300 are a set of flexible bags (not shown) used to contain allof the processing fluids and waste fluids. These bags are connected tothe fluid pathway (not shown) via the needleless connections 910. Priorto connection of the fluid reservoir 300 to the upper housing 200 of thetissue engineering module 118, connections 910 are also used to installfluids into each bag, as well as provide access for adding finalcomponents (such as autologous serum). In order to ensure that thefluids contained within the bags in the fluid reservoir 300 remainviable for the extended periods required for cell culture and tissueengineering, the fluid reservoir 300 has been designed to allow forreduced temperature operation as compared with the remainder of thetissue engineering module 118, which typically operates at 37° C. Thereduced temperature within the fluid reservoir 300 is attained via atemperature controller, such as Peltier1M cooling elements on the baseof the system bay (not shown), which protrude upwardly into the fluidreservoir 300 through holes 912 to provide local cooling to the bags.The bag temperature sensor (not shown) resides on the base of the systembay (not shown) and protrudes through a hole 914 in the fluid reservoir300 to provide the control feedback necessary for temperature control.Local cooling within the overall module 118 for the tissue engineeringsystem 100 has the advantage of minimizing the power required forcooling and also minimizing problems associated with condensation. Toensure that there is adequate insulation for the inner fluid reservoirbags, the fluid reservoir 300 is constructed to be a double walledreservoir 916. An air space is present between the two walls therebyimproving the insulation properties of the reservoir 300 while,preferably, maintaining optical clarity for inspection. Through activecooling of the fluid reservoir 300, the contents remain in arefrigerated state thereby minimizing or eliminating operatorintervention to replace fluids that would otherwise expire if maintainedcontinuously at 37° C. Latches 918 on the corners of the fluid reservoir300 provide the connection points that assemble the fluid reservoir 300to the upper housing 200 of the tissue engineering module 118.

FIG. 10 shows a flow control valve housing 1000 of the upper housing200, which is used as the main fluid pathway tube interconnect and fluiddirection interface between the chamber assemblies 500, 600 and 700 andthe fluid reservoir 300. The flow control valve housing 1000 isconstructed as a molded plastic component that makes connections to thefluid reservoir 300 through the series of cannula connections 1010.Fluid control is obtained via flow control valves 212 that are installedinto the flow control valve housing 1000. The flow control valve headers1012 and 1014 provide a common fluid connection between the flow controlvalves 212 and enables the internal fluid pathway connections betweenthe chamber assemblies 500, 600 and 700 and the fluid reservoir 300.This housing 1000 and related vertical plates (not shown) dramaticallysimplifies the internal tubing complexity for the fluid managementsystem used within the tissue engineering module 118.

FIG. 11 shows an exploded view of the tissue engineering module 118 ofFIG. 4 with the fluid reservoir 300 of FIG. 9. FIG. 11 shows the twomain components of the tissue engineering module 118 in the final stageof installation; the upper housing 200 and the lower fluid reservoir300. To summarize, the upper housing 200 contains the installed tissuedigest bioreactor 510 within a protective containment unit 520; theproliferation chamber assembly 600; the installed differentiationbioreactor 710 within the protective containment unit 720 or the celltherapy bioreactor 810 within the protective containment unit 820; andthe flow control valve housing 1000. The tissue engineering module 118contains internal crossflow cell concentrator (not shown) andinterconnect tubing and valves to complete the fluid handling system(not shown). Connection of the upper housing 200 to the fluid reservoir300 automatically engages a series of fluid connectors that enable fluidcommunication between the two components 200 and 300. Both components200 and 300 are held together by molded latches (not shown) on the upperhousing 200 and molded latches 918 on the fluid reservoir 300.

FIG. 12 shows a scheme of a general methodology for clinical celltherapy and tissue engineering using the tissue engineering module 118of FIG. 3, operating in the tissue engineering system 100 of FIGS. 1 and2, and autologous cartilage tissue engineering as a representativeexample. In such an example, cells (i. e. chondrocytes) are obtainedfrom a surgical biopsy of a patient and placed in the tissue digestionbioreactor 510 of the tissue digestion chamber assembly 500. The tissuedigestion chamber assembly 500 is engaged with the tissue engineeringmodule 118 containing the proliferation chamber assembly 600 and productchamber assembly 700. A central microprocessor is present within thetissue engineering system and controls and customizes the internalenvironment of the bioreactor/chambers, and hence facilitates tissuegrowth therein, resulting in the stimulation of cell growth andsubsequent matrix expression to generate an implant. Sensors within thebioreactor provide feedback to the microprocessor to ensure that thecells are seeded, expanded and differentiated in a desired andcontrolled manner to provide an autologous tissue implant. Once theimplant is generated, the product chamber assembly 700 is removed fromthe module 118 and transported to the operating room for surgicalimplantation into the patient. The present system provides anadvantageous way to provide autologous tissue engineered implants in asterile, safe, convenient and efficacious manner. Furthermore, theability to prepare tissue engineered implants in a clinical settingallows considerable flexibility in the locations for undertaking thetissue engineering process. While the system can be used in acentralized location, the design and automated operation of the systemenables clinical use at regional centers. Such widespread availabilityprecludes the transportation of biological material to and fromcentralized cell/tissue processing facilities, thereby improving thecost effectiveness and efficiency of the tissue engineering processwhile avoiding shipment, tracking and regulatory complications.

FIG. 13 illustrates an embodiment of a fluid flow schematic in which thebioreactors/chambers of FIGS. 5 and 6, and either 7 or 8 may beemployed. A tissue digestion bioreactor 510 is present that accommodatesa tissue biopsy. A proliferation chamber assembly 600 is present that isconfigured to accept cells from the tissue digestion bioreactor 510 andallows seeding of the proliferation surface 610. Bubble traps within thetissue digestion bioreactor 510 remove air bubbles from the input lineto the proliferation chamber assembly 600 and therefore prevents thesebubbles from entering the proliferation chamber assembly 600 andpotentially compromising localized cell populations. A cell washingreservoir 220 is present to accept the expanded cell numbers from theproliferation chamber assembly 600 and to serve as a temporary holdingcontainer during a cell washing and cell concentration procedureperformed with the aid of a cross flow filtration module 222. One ofseveral alternative product bioreactors (e.g. cell therapy bioreactor810, differentiation/tissue formation bioreactor 710, a multi-implantbioreactor 1310, or a cell matrix implant bioreactor 1312) is alsopresent and is configured to accept the cells from reservoir 220 afterthe washing and concentration step.

Tissue engineering reagents (i.e. media, enzyme solutions, washingsolutions, etc.) and waste fluids are stored in a fluid reservoir 300such as that shown in FIG. 9. Fluid flow through the system is directedby the operation of a fluid pump 224, flow control valves 212 a-212 gand 212 r-212 v according to control inputs from a centralmicroprocessor. Air filters allow the transfer of air into or out of thesystem as required during operation without compromising systemsterility. Furthermore, in-line gas exchange membranes (not shown) maybe deployed at various locations within the fluid flow paths tofacilitate the control of dissolved gases in the culture medium.

In one non-limiting example of the system operation, a tissue biopsy isinserted into the tissue digestion bioreactor 510 of the tissuedigestion chamber assembly 500. A digestion medium containing enzymes ispumped into the tissue digestion bioreactor 510 from the fluid reservoir300 to initiate the digestion process. The digestion medium may becontinuously or periodically re-circulated via pump 224, thereby keepingthe released cells in suspension and maximizing reagent exposure to thebiopsy. Introduction of a proliferation culture medium from the fluidreservoir into the top of the tissue digestion bioreactor 510 transfersthe cell suspension to the proliferation chamber assembly 600 andsimultaneously dilutes the enzyme solution to a concentration that istolerable for cell proliferation. The transfer of partially digestedtissue out of the digestion bioreactor 510 is precluded by port filterthat is sized to allow passage of disassociated cells and retention ofcell aggregates. Cells generated from the biopsy digestion process arehomogeneously distributed throughout the proliferation chamber assembly600 either by the recirculation of the cell suspension via theactivation of valves 212 and the pump 124, or by the automatedapplication of gentle shaking of the proliferation chamber assembly 600.

Following a quiescent period to allow attachment of the cells to theproliferation surface 610, the proliferation medium is periodically orcontinuously replaced with fresh proliferation medium from the fluidreservoir 300. During a medium replacement step, the supply of freshmedium from the fluid reservoir 300 is balanced by the discharge ofwaste fluid to a waste container in the fluid reservoir 300 via valve212 g.

Once the cell culture approaches confluence, the media within theproliferation chamber assembly 600 is evacuated into the waste containerwithin the fluid reservoir. In this process, the removal of fluid fromthe proliferation chamber assembly 600 is balanced by incoming sterileair delivered via an air filter or by incoming PBS wash solution fromthe fluid reservoir 300.

The cells are subsequently released from the proliferation surface 610through an automated sequence, such as the delivery of enzymes (forexample trypsin) and the timed recirculation of the cell suspension orthe timed application of impact or agitation to the bioreactor via animpact drive. In order to remove the enzymes and to collect the cells ina relatively small volume of medium for subsequent transfer to aselected product bioreactor (710, 810, 1310, or 1312) of the productchamber assembly, the cell suspension is transferred from theproliferation chamber assembly 600 to the cell washing reservoir 220.The cell suspension is then continuously recirculated via valves 212 andpump 224 through the cross-flow filtration module 222. The membrane inthe cross-flow filtration module 222 prevents the loss of cells butallows a certain percentage of media (permeate) to be removed via valve212 g to the waste container in the fluid reservoir 300. The result is areduction of the suspension volume and/or dilution of any enzymespresent, provided the removal of permeate is compensated by the supplyof fresh medium from the fluid reservoir 300. The continuous flowreduces the potential for cells to become entrapped within the membraneof the cross-flow filtration module 222.

Cell delivery to the product bioreactor is achieved by transferring thewashed cells from the reservoir 220 via the valves 212 and pump 224.Following cell transfer to the product bioreactor, fresh media may beintroduced into the product bioreactor through the operation of pump224. During biological processing, the medium is periodically orcontinuously replaced with fresh medium from the fluid reservoir 300.During a medium replacement step, the supply of fresh medium from thefluid reservoir 300 is balanced by the discharge of waste fluid to awaste container in the fluid reservoir via valve 212 g. In between themedium replacement steps, the fluid within the product bioreactor iscontinuously or periodically recirculated under the control of pump 224and valves 212. In order to ensure that environmental conditions withinthe different bioreactors promote normal cellular activity, conditionsare monitored and controlled for the period necessary for the successfulcollection of expanded cells in the case of cell therapy or formation ofone or more tissue constructs in the case of tissue engineering. Oncethe collection of cells or formation of tissue implants is complete, theproduct bioreactor is removed and transported to the operating room forsubsequent clinical use

It should be noted that the system/module of the invention is notlimited to a particular type of cell or tissue. For example, a skeletalimplant may be prepared for use in the reconstruction of bone defects.In this application, bone marrow could be used as the source of theprimary and/or precursor cells required for the tissue engineeringprocess. Accordingly, there is no requirement to perform tissuedigestion; hence, the bioreactor chamber assembly may be of the typethat only supports proliferation and differentiation. Depending on theavailable cell population and the required size of the implant, evenproliferation may not be required. In this case, the configuration ofthe bioreactor chamber assembly may be directed to the single stage ofcell differentiation and ongoing tissue formation. The final tissueconstruct could be comprised of an implantable scaffold, which may becomposed of a bone biomaterial such as Skelite™, with active bone cellslining the open pores of the scaffold and actively laying down newmineralized matrix (osteoid). Such an implant would be quicklyintegrated at the implant site thereby accelerating the recoveryprocess.

When two or more chamber assemblies are used in the module, the chamberassemblies may be independently operable or co-operatively operable. Forexample, the chamber assemblies may be operatively connected such thatthere is an exchange of fluids, cells and/or tissues from chamber tochamber or the chamber assemblies may operate independent of oneanother. The chamber assemblies may be connected via at least one of apassageway, tubing, connector, valve, pump, filter, fluid access port,in-line gas exchange membrane, and in-line sensor.

The tissue engineering system of the present invention is designed toperform activities under aseptic operating conditions. The system isfully automated, portable, multifunctional in operation andperforms/provides without being limited thereto, one or more of thefollowing:

-   -   sterile reception/storage of tissue biopsy;    -   automated monitoring of digestion process    -   digestion of biopsy tissue to yield disassociated cells;    -   cell sorting and selection, including safe waste collection;    -   cell seeding on or within a proliferation substrate or scaffold    -   proliferation of cells to expand cell populations;    -   cell washing and cell collection;    -   cell seeding on or within a tissue engineering scaffold,        membrane and/or matrix;    -   cell differentiation to allow specialization of cellular        activity;    -   tissue formation;    -   mechanical and/or biochemical stimulation to promote tissue        maturity;    -   harvesting the tissue engineered constructs/implants for        reconstructive surgery; and    -   storage and transportation of implantable tissue.

The tissue engineering system of the present invention may bepre-programmed to perform each of the above noted steps and/or othersteps, individually, sequentially or in certain predetermined sequencesor partial sequences as desired and required. Furthermore, each of thesesteps, or any combination thereof, are accomplished within one or morechamber assemblies on a tissue engineering module. In operation, thetissue engineering system is pre-programmed and automatically controlledthus requiring minimal user intervention and, as a result, enhances theefficiency and reproducibility of the cell culture and/or tissueengineering process while minimizing the risks of contamination.Therefore, in one example, the automated tissue engineering system ofthe present invention is capable of multi-functionally carrying out allof the steps of a biopsy tissue digestion to yield disassociated cells,subsequent cell seeding on a proliferation substrate, cell numberexpansion, controlled differentiation, tissue formation and/orproduction of a tissue implant within a single system.

The tissue engineering system and tissue engineering module is not to belimited to tissue engineering per se. The system and module can beutilized, for example, for cell therapy. Therefore, the applicability ofthe system and module of the present invention ranges from tissueengineering; to the formation of cells and/or tissues on and/or withinat least one scaffold, membrane and matrix; to, simply, the expansion ofcells for cell therapy applications. It is noted that thescaffolds/membranes/matrices can be any suitable shape, such ascontoured, circular, have an irregular perimeter. The term “cell matriximplant” used herein is understood to encompass cells within and/or on ascaffold, matrix, and/or membrane, such as, and without being limitedthereto, a pre-tissue.

Cells and tissues may be selected from, and without being limitedthereto, non-cartilage tissue, such as cardiac tissue, vascularimplants, and skin grafts, and skeletal tissues such as bone, cartilage,tendon, disc, related bone and cartilage precursor cells, andcombinations thereof. More specifically, cells suitable for use inchamber assemblies, module and system of the invention are selected frombut not limited to the group consisting of embryonic stem cells, adultstem cells, osteoblastic cells, pre-osteoblastic cells, chondrocytes,nucleus pulposus cells, pre-chondrocytes, skeletal progenitor cellsderived from bone, bone marrow or blood, including stem cells, andcombinations thereof. The cells or tissues may be of an autologous,allogenic, or xenogenic origin relative to the recipient of an implantformed by the cell culture and tissue engineering functions of theinvention. It is also understood that the term tissues, as used herein,is not to be limited only to connective tissues but can include avariety of tissues such as, and without being limited thereto, cardiactissue, vascular implants, and skin grafts.

The chamber assemblies of the present invention may provide anenvironment for at least one of the following selected from the groupconsisting of storage of tissue biopsy, digestion of tissue biopsy, cellsorting, cell washing, cell concentrating, cell seeding, cellproliferation, cell differentiation, cell storage, cell transport,tissue formation, implant formation, storage of implantable tissue andtransport of implantable tissue.

The sensors used herein, such as, for example, confluence sensors, mayhave ability to monitor the specific performance of cellpopulations/tissue in said at least one chamber assembly from variousdonors and thereby, allow the system to accommodate for the requirementsof cells/tissue of individual donors in said at least one chamberassembly. For example, as a result of these sensors, the system has theability to adapt to the needs of specific cells/tissues from differentdonors. For instance, cells from an older donor may grow at a slowerrate compared to a younger donor, therefore, the sensors would permitthe system to adjust accordingly to permit longer growth times.

In addition, the tissue engineering module of the present invention canhave at least one additional chamber assembly that can share a commonprocess with an existing chamber assembly such that the additionalchamber assembly can be removed in order to provide analysis and/orevaluation of the contents of the chamber that parallels the contents ofthe existing chamber. The contents may be media, tissue and/or cells.

The advanced tissue engineering system of the present invention hasseveral advantages compared to methods and systems of the prior art. Inparticular, the turn-key operation of the device enables complex tissueengineering procedures to be performed under automated control in theclinic, thereby precluding the need to transport cells to centralizedfacilities for biological processing. The system is simple to use andobviates the existing time consuming and expensive manual human tissueculture procedures which can lead to implant contamination and failure.The tissue engineering modules and associated subsystem assemblies maybe customized for the type of cell or tissue to be cultured and may befabricated from any suitable biocompatible and sterilization tolerantmaterial. The entire tissue engineering module or specific componentsthereof are replaceable and may be considered disposable. The tissueengineering module may be provided in a single-use sterile package thatsimplifies system set-up and operation in clinical settings. In otherembodiments, any components such as the tissue digestion chamberassembly and product chamber assembly as well as the housing and thefluid reservoir can be provided separately packaged for use as a kit. Inembodiments of the invention, the tissue digestion chamber assembly andthe product chamber assembly with a selected bioreactor therein, may beprovided separately packaged and as such can be provided as a kit to beused with a tissue engineering module. The proliferation chamberassembly in aspects is fabricated already attached to the housing of thetissue engineering module. All detachable aspects of the tissueengineering module are designed to ensure that assembly can only be donewith the correct orientation and once assembled is essentiallytamperproof.

It is understood by those skilled in the art that the tissue engineeringmodule and device of the present invention can be fabricated in varioussizes, shapes and orientation. The device can be fabricated toincorporate a single tissue engineering module or multiple modules invertical or horizontal formats. Accordingly, the subassemblies can bemade to correspond to the spatial format selected for the tissueengineering device. As such, different types of tissue engineering canbe simultaneously conducted in a single device with each tissueengineering sequence being automatically monitored and controlled on anindividual basis. It is also within the scope of the invention to have aplurality of automated tissue engineering systems operating andnetworked under the control of a remote computer.

The present invention is an improvement to the Applicant's automatedtissue engineering system described in International Patent ApplicationNo. WO 03/0872292. The improvements to the advanced tissue engineeringsystem of the present invention are generally discussed below.

In one aspect of the invention, the tissue engineering module stillcontains multiple bioreactors provided within chamber assemblies toallow multistage processing (digest/proliferation/differentiation);however, there are new aspects to the tissue engineering module asfollows:

-   -   The flow pathway of the advanced system is comprehensively        revised reducing the number of valves. This can be achieved        through an innovative use of check valves with specific cracking        pressures;    -   Since the advanced system can be comprised of a series of        disposable components assembled in the clinic at the time of use        (disposable tissue engineering module, fluid reservoir, chamber        assemblies (tissue digestion, proliferation and one of four or        more product chambers)), the tissue engineering module can        include assembly integrity sensors that monitor that all parts        are present and are connected together correctly;    -   Predictive software can be included in combination with        biosensor feedback to enhance control over the bioprocessing of        the advanced system enabling the implantation surgery to be        forecast in advance;    -   The advanced system can accommodate the use of autologous        (patient) serum as well as autologous cells, thereby minimizing        risk;    -   The advanced system can allow for multiple sample ports for the        removal of media and/or cell and media samples;    -   In addition, in the advanced system, ports can be available to        allow mid process loading of additional media and/or additives,        in the event this is necessary for certain clinical activities;        and    -   In addition to the assessment of cell vitality and cell number        as part of a quality control kit, the advanced tissue        engineering system can support the innovative use of assays in        the form of microarrays and protein expression arrays. This is        facilitated by the fact that a user may have access to the        various components of the module such as for example the        proliferation chamber assembly. In this manner, cells may be        tested for expression of certain genes and proteins at various        steps during the processing and operation of the system.

The Reservoir

-   -   The fluid reservoir is installed as a separate unit (as shown in        FIG. 11) and, in one embodiment, fluid connections are provided        via connectors, such as an array of needleless ports, on the top        surface that engages with mating connectors present in the upper        housing of the tissue engineering module. The connectors and        mating connectors are in fluid communication with one another;    -   The fluid reservoir is optionally pre-filled;    -   The fluid reservoir is optionally pre-sterilized;    -   The fluid reservoir may be structurally rigid for ease of        handling;    -   The attachment of the fluid reservoir to the tissue engineering        module may be via a one-way snap-on connection. Once attached,        the reservoir cannot be detached; thereby, precluding        potentially hazardous (and contra-indicated) re-use;    -   The fluid reservoir may be clear for inspection of contents and        to allow visible confirmation of additive loading prior to        connection to the cassette;    -   The fluid reservoir may have open “windows” in the base to        enable thermal union with Peltier (or similar) cooling members        that emerge from the base of the bay present on the instrument;    -   The fluid reservoir may be designed with twin walls to maximize        the insulation properties when operated at 4° C. and the        remainder of the instrument at 37° C.

The Bioreactors

The bioreactor design is significantly different than the Applicant'searlier PCT application. While the basic internal working of the tissuedigestion bioreactor, the proliferation bioreactor, and thedifferentiation bioreactor for implant formation are per the Applicant'searlier International Patent Application No. WO 03/0872292, the designhas been improved. In one particular embodiment, a novel design for theprovision of a double containment for selected bioreactors has beenimplemented to improve and maintain aseptic conditions during transportof these bioreactors to or from the clinic or operating room. Thechamber assembly for the tissue digestion bioreactor and/or the productbioreactor (differentiation bioreactor) comprises an outer protectiveunit that houses the selected bioreactor therein. The outer protectiveunit comprises a unit lid and unit base. Engaged within the outerprotective unit is a desired bioreactor that comprises a bioreactor lidand bioreactor base. The bioreactor lid may be supported and engageablewith a portion of the unit lid by needleless injectors.

Chamber Assembly—with Tissue Digestion Bioreactor

-   -   The chamber assembly having a tissue digestion bioreactor        therein is designed to accept a tissue biopsy (for example but        not limited to a cartilage biopsy, in other aspects may be        loaded with cells) and facilitates directed flow as outlined in        the Applicant's International Patent Application No. WO        03/0872292. In one embodiment, the tissue digestion bioreactor        within the assembly is formed with the bioreactor base chamber        containing an integral lower tubing connection such that all the        flow port connections occur at the top. This enables connection        to a top manifold through sterile needleless connections (FIGS.        5B and 5C).    -   In addition the tissue digestion bioreactor chamber assembly may        be produced with two “containment layers” whereby the tissue        digestion bioreactor and connection ports are loaded into an        outer protective containment unit with a further set of        connection ports (Figures SB and SC). The ports are designed to        allow aseptic docking once the protective port covers (tabs or        adhesive labels) are removed. This twin level of protection        provides important added security to prevent inadvertent        contamination as the tissue digestion bioreactor within the        chamber assembly is being transported from the site of biopsy        collection (e.g. operating room) to the location of the advanced        tissue engineering system (e.g. clinical lab).        Chamber Assembly—with a Proliferation Bioreactor

The proliferation bioreactor is similar to the “s-channel” bioreactorshown in Applicant's International Patent Application No. WO 03/0872292.However, there are several changes made thereto that provide importantimprovements:

-   -   One embodiment of the layout of the proliferation bioreactor is        in a race-track configuration (FIG. 6) that is similar but not        limited to the letter “C”. This allows the inlet and outlet to        be placed at the center of the tissue engineering module,        thereby facilitating tubing connections.    -   The race-track has inlet and outlet ports that enter the        proliferation chamber with a duct that increases in width as the        chamber is approached. This reduces the streamlining of the flow        and allows a more uniform fluid distribution into and out of the        chamber.    -   The volume of the proliferation bioreactor was considered in        terms of the resulting dilution that occurs when the incoming        cell suspension released from the digest bioreactor is mixed        with incoming media to fill the proliferation chamber. It was        found that residual enzymes initially used in the digestion        process do not need to be physically removed or deactivated (to        preclude cell complications during proliferation) when dilutions        of for example about 10:1 occurs during the loading of the        proliferation chamber.    -   The height of the proliferation bioreactor can be optimized to        obtain an intermediate height between a low height that allows        air bubbles to bridge between the top surface and the active        cell surface (causing cell necrosis), and a higher height where        the fill volume is excessive and air removal is problematic.    -   The proliferation bioreactor can optionally include flow        interrupters that deliberately cause controlled turbulence along        the length of the proliferation surface. These interrupters        would be placed perpendicular to the flow as irregularities in        the ceiling of the proliferation chamber. The objective is to        cause controlled mixing along the length of the proliferation        surface so that free cells (particularly post release after        confluence detection) remain in suspension and can be moved        efficiently toward the outlet.    -   The proliferation bioreactor may optionally include a slight        elevation change from inlet to outlet (cork-screw style or        similar to a spiral ramp) to enable the more complete exhaust of        all contents. This elevation change would be accomplished with        both the floor and ceiling of the cavity decreasing in elevation        at the same rate, thereby maintaining a consistent interior        height.    -   The proliferation bioreactor includes sensors to monitor the        onset of cell confluence. In one aspect, sensor electrodes        reside on the proliferation surface and are exposed to the media        to monitor the changes in impedance that occurs with increasing        cell growth.    -   The proliferation bioreactor optionally includes a vibratory        element that facilitates cell release from the proliferative        surface of the chamber. This element is mounted directly in the        chamber.    -   In addition, multiple proliferation bioreactors may be        incorporated.        Chamber Assembly—with Product (i.e. Implant) Bioreactor    -   Four different product bioreactors may be selected for use in        the product chamber assembly (identified generically as        differentiation bioreactors in the Applicant's International        Patent Application No. WO 03/0872292). The product formats are:        -   Cell therapy bioreactor—In one representation, there is a            vial contained within the overall bioreactor (FIG. 8C) where            the vial enables cell sedimentation. After cell            sedimentation the design of the cell therapy bioreactor            provides for the removal of the media supernatant leaving            the concentrated cells in a small cone at the base of the            vial. This approach provides a vial with concentrated cells            as is now provided by centralized cell therapy providers.            With this automated technique, additional use of a            centrifuge to concentrate the cells at the end of the            process as per conventional manual techniques is not            required.        -   Single (TE) tissue engineered bioreactor—The bioreactor            facilitating all ports at the top for easy connection with            the system via needleless connectors (FIGS. 7B and 8B). The            approach to the formation of the tissue engineered implant            is similar to that described in Applicant's International            Patent Application No. WO 03/0872292)        -   Multiple (TE) tissue engineered implants—This is similar to            that defined in the Applicant's International Patent            Application No. WO 03/0872292.        -   Cell matrix implant bioreactor—This is a hybrid of cell            therapy and tissue engineering where cells are cultured for            a short period on a matrix to allow attachment but minimal            tissue formation. The cell matrix may be flexible. This            technique has been references as MACI approach (matrix            induced autologous chondrocyte implantation).        -   The product bioreactor (in any of the aforementioned            formats) also benefits from the twin “containment layers”            (as shown in FIGS. 7A, 7B, 8A, and 8B) as substantially            employed for the tissue digestion bioreactor. In the case of            a product bioreactor, the value of the twin layers (i.e. the            outer containment unit) is to allow the bioreactor contained            therein to be disassembled in a sequence consistent with            operating room practice. That is to say that the exterior            can be removed and the interior parts handled while            maintaining aseptic practices.

Other Components

-   -   Beyond the different bioreactor chamber assemblies that        incorporate the double containment system, a high efficiency        cross-flow filter can be implemented to enable cell        concentration post proliferation collection. This component        eliminates centrifugation. This component is shown in the flow        diagram (FIG. 13).    -   In aspects, a space and cost efficient valve manifold has been        designed that provides the fluid management described in the        Applicant's International Patent Application No. WO 03/0872292        while also providing the structural support for the valve array,        the critical fluid interconnects between the valves the        bioreactors and the fluid reservoir, and the attachment points        for the tissue engineering module latches.

A cell collection reservoir has been included in the design as a stagingarea and to allow for warming of fluid from the reservoir prior toinfusion into the different chamber assemblies.

Although preferred embodiments have been described herein, it isunderstood by one of skill in the art that variations may be madethereto without departing from the spirit of the invention.

What is claimed is:
 1. A product chamber assembly, comprising: abioreactor; a set of flexible bags that are used to contain processingfluids and for automated flow to and from the bioreactor, wherein atleast one flexible bag operates at reduced temperature from thebioreactor, and wherein at least one flexible bag is operationallyengaged with a collection reservoir; and at least one biosensor for themonitoring of parameters of the flexible bags and cells within thebioreactor, the at least one biosensor relaying variable parameterinformation to a microprocessor to process the parameters anddynamically adjust and adapt to specific needs of cells during cellproliferation.
 2. The product chamber assembly of claim 1, wherein theset of flexible bags and the bioreactor are connected via at least oneof a passageway, tubing, connector, valve, pump, filter, fluid accessport, in-line gas exchange membrane, and in-line sensor.
 3. The productchamber assembly of claim 1, wherein the product chamber assemblyprovides an environment for at least one of the following: cell sorting,cell washing, cell concentrating, cell seeding, cell proliferation, cellstorage, and cell transport.
 4. The product chamber assembly of claim 1,wherein the bioreactor is a cell therapy bioreactor.
 5. The productchamber assembly of claim 4 wherein the cell therapy bioreactor collectsand holds proliferated cells for cell therapy applications.
 6. Theproduct chamber assembly of claim 1, wherein the at least one biosensorhas the ability to monitor the specific performance of cells in thebioreactor and thereby, allow the system to accommodate for therequirements of cells of an individual in the product chamber assembly.7. The product chamber assembly of claim 1, wherein the product chamberassembly is portable.
 8. The product chamber assembly of claim 1,wherein the bioreactor is removable from the product chamber assembly.9. The product chamber assembly of claim 1, wherein the bioreactorfurther comprises at least one of a gas permeable membrane, flowinterrupters, and vibratory elements.
 10. A product chamber assemblycomprising: a bioreactor; a set of flexible bags that are used tocontain processing fluids and for automated flow to and from thebioreactor, wherein at least one flexible bag is in fluid communicationwith the bioreactor; a flow control valve housing comprising one or morecontrol valves; and at least one biosensor for monitoring parameters ofthe flexible bags and cells within the bioreactor, the at least onebiosensor relaying variable parameter information to a microprocessor toprocess the parameters and dynamically adjust and adapt to specificneeds of the cells.
 11. The product chamber assembly of claim 10,wherein the bioreactor is a cell therapy bioreactor.
 12. The productchamber assembly of claim 10, further comprising a collection reservoir.13. The product chamber assembly of claim 10, further comprising a fluidpump.
 14. The product chamber assembly of claim 10, wherein the at leastone flexible bag is fluidly connected to the bioreactor via a needlessconnection.
 15. The product chamber assembly of claim 10, wherein theflow control valve housing is a fluid pathway tube interconnect andfluid direction interface between the bioreactor and the at least oneflexible bag.